This invention relates to the in vitro culture of living cells, such as animal cells, to achieve reproduction and growth of the cells to a desirably high cell number, and more particularly to methods and apparatus for the preparation of high cell number cell masses for seeding production-scale culture systems.
The in vitro culture of animal cells has long been in use for a variety of purposes, and in recent years has received considerable attention and achievement as a means for production and recovery of cell-manufactured proteins of established or potential therapeutic and/or diagnostic utility, be it through culture of naturally-occurring, protein-producing animal cells, or culture of protein-producing hybrids formed from such animal cells, or culture of animal host cells which have been transformed, via recombinant DNA technology, using heterologous genes coding for a particular protein product.
For most such processes, relatively large-scale culture systems are desirable as a means for meeting production demands for the protein and/or for decreasing the per unit production cost of the protein. As is well-known, it is neither economic nor generally even feasible to initiate such culture processes utilizing only a small number of cells. As a consequence, it is routinely necessary to first separately produce a homogeneous cell mass of sufficiently high cell number so as to provide an initial inoculum of cells to the production culture system which insures viability therein and results in economic production of cell-manufactured proteins.
To this end, a small initial charge of cells and an appropriate quantity and type of culture medium are introduced into a suitable small volume vessel such as a tissue culture (T) flask, spinner flask, roller bottle or the like, in the presence of a suitable gaseous environment for providing, e.g., a mixture of oxygen and carbon dioxide to the cells. In this small-scale environment, a mass of low cell number can retain viability and reproduce and grow to a higher cell density until confluency is attained.
If a suitably high cell number can be directly produced in this manner, the cells must then be removed from the flask or bottle and then inoculated, optionally as a suspension in fresh culture medium, into the production culture unit, an operation which can be difficult and time-consuming and, most importantly, must be carried out under strictly sterile conditions lest the entire inoculum be contaminated.
As is more often generally the case, the increase in cell number achieved in the small flask or bottle is not sufficiently great to permit the cells to be directly introduced into the production culture unit, yet the original flask or bottle is too small to accommodate further medium, growth and reproduction. In such circumstances, the cells are transferred to a suitably larger flask or bottle for further introduction of medium and further growth and reproduction to an increased cell number. For many cell lines, a number of such transfers to increasingly larger capacity vessels is needed before a viable cell mass of sufficiently high cell number is achieved which can be inoculated into the production culture unit.
With each transfer of a cell mass from one preliminary growth vessel to another and eventually to the production culture unit, the risk of contamination of the inoculum exists. If such contamination occurs, say, in the later stages of transfer, the entire process must be repeated from the beginning thereby greatly increasing the time and cost involved in obtaining a suitable inoculum for the production culture unit and greatly increasing the time and cost involved in culturing cells for recovery of their manufactured proteins.
The primary object of the present invention is to provide a means for growing up cells to a cell number suitable for introduction into a production culture system, under conditions which provide a sufficiently small-scale starting environment for insuring viability of the cells, an increasingly larger-scale environment as necessary, and a means for eventual introduction of the high cell number mass to a culture unit, without risk of transfer contamination and in an economic and time-conserving manner.